Oligonucleotide sequences targeting transcription factor TSC22D4 for the treatment of insulin resistance

ABSTRACT

The present invention relates to oligonucleotide inhibitors of the TSC22D4 activity or expression and their uses for the prevention, treatment, and/or regulation of insulin resistance, metabolic syndrome and/or diabetes and/or for improving insulin sensitivity in a mammal.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a National Stage Application of InternationalApplication Number PCT/EP2016/053050, filed Feb. 12, 2016; which claimspriority to European Patent Application No. 15160259.6, filed Mar. 23,2015; both of which are incorporated herein by reference in theirentirety.

The Sequence Listing for this application is labeled“SeqList-15May19-ST25.txt”, which was created on May 15, 2019, and is 2KB. The entire content is incorporated herein by reference in itsentirety.

The present invention relates to oligonucleotide inhibitors of theTSC22D4 activity or expression and their uses for the prevention,treatment, and/or regulation of insulin resistance, metabolic syndromeand/or diabetes and/or for improving insulin sensitivity in a mammal.

BACKGROUND OF THE INVENTION

In humans, a combination of excessive lipid storage and decreasedremoval leads to overweight and associated co-morbidities, includinginsulin resistance, cardiovascular complications, and dyslipidemia(Langin D. In and out: adipose tissue lipid turnover in obesity anddyslipidemia. Cell Metab. 2011 Nov. 2; 14(5):569-70), now affecting morethan 1.5 billion people worldwide (Finucane M M, et al. National,regional, and global trends in body-mass index since 1980: systematicanalysis of health examination surveys and epidemiological studies with960 country-years and 91 million participants. Lancet. 2011 Feb. 12;377(9765):557-67). Indeed, insulin resistance represents the corecomponent of the so-called metabolic syndrome, ultimately leading to thedevelopment of metabolic dysfunction, such as glucose intolerance,pancreatic beta cell failure, and eventually type 2 diabetes.

Impaired insulin secretion (beta-cell), increased hepatic glucoseproduction (liver), and decreased peripheral (muscle) glucoseutilization constitute the traditional primary defects responsible forthe development and progression of type 2 diabetes mellitus. Beta-Cellfailure, ultimately leading to decreased insulin secretion, is now knownto occur much earlier in the natural history of type 2 diabetes thanoriginally believed. Additionally, a better understanding of thepathophysiology of type 2 diabetes reveals other etiologic mechanismsbeyond the classic triad, now referred to as the ominous octet. Inaddition to the beta-cell, liver, and muscle, other pathogenicmechanisms include adipocyte insulin resistance (increased lipolysis),reduced incretin secretion/sensitivity (gastrointestinal), increasedglucagon secretion (alphacell), enhanced glucose reabsorption (kidney),and central nervous system insulin resistance resulting fromneurotransmitter dysfunction (brain). Currently, the management of type2 diabetes focuses on glucose control via lowering of blood glucose(fasting and postprandial) and hemoglobin A(1c). However, the goal oftherapy should be to delay disease progression and eventual treatmentfailure. Treatment should target the known pathogenic disturbances ofthe disease (i.e., reducing the deterioration of beta-cell function andimproving insulin sensitivity). In recent years, treatment strategieshave focused on the development of novel therapeutic options that affectmany of the defects contributing to type 2 diabetes and that providedurable glucose control through a blunting of disease progression.Optimal management of type 2 diabetes should include early initiation oftherapy using multiple drugs, with different mechanisms of action, incombination (DeFronzo R A. (Current issues in the treatment of type 2diabetes. Overview of newer agents: where treatment is going. Am J Med.2010 March; 123(3 Suppl):S38-48).

Especially the insensitivity of major metabolic organs against insulinaction, including the liver, skeletal muscle and adipose tissue,substantially contributes to disease progression and the ultimate needfor pharmacologic intervention to prevent diabetic late complications.Thus, efficient and safe insulin sensitization remains an attractivetarget and aim in anti-diabetic therapy.

Transcriptional co-factor complexes have been identified as importantcheckpoints in the coordination of metabolic programs in varioustissues, including liver and white adipose tissue (WAT) (for a review,see Sommerfeld A, Krones-Herzig A, Herzig S. Transcriptional co-factorsand hepatic energy metabolism. Mol Cell Endocrinol. 2011 Jan. 30;332(1-2):21-31).

Kester H A, et al. (in: Transforming growth factor-beta-stimulatedclone-22 is a member of a family of leucine zipper proteins that canhomo- and heterodimerize and has transcriptional repressor activity. JBiol Chem. 1999 Sep. 24; 274(39):27439-47) describe thatTGF-beta-stimulated clone-22 (TSC-22) encodes a leucinezipper-containing protein that is highly conserved during evolution.

Furthermore, Jones et al. (in Jones, A., et al., Transforming growthfactor-beta1 Stimulated Clone-22 D4 is a molecular output of hepaticwasting metabolism. EMBO Mol Med. 2013 February; 5(2):294-308) describethat as a molecular cachexia output pathway, hepatic levels of thetranscription factor transforming growth factor beta 1-stimulated clone(TSC) 22 D4 were increased in cancer cachexia. Mimicking high cachecticlevels of TSC22D4 in healthy livers led to the inhibition of hepaticVLDL release and lipogenic genes, and diminished systemic VLDL levelsunder both normal and high fat dietary conditions. Therefore, hepaticTSC22D4 activity may represent a molecular rationale for peripheralenergy deprivation in subjects with metabolic wasting diseases,including cancer cachexia.

Kulozik, Ph., et al. (Hepatic deficiency in transcriptional co-factorTBLI promotes liver steatosis and hypertriglyceridemia. 2011 Cell Metab.13: 389-400) describe that the impaired hepatic expression oftranscriptional cofactor transducin beta-like (TBL) 1 represents acommon feature of mono- and multigenic fatty liver mouse models. Theliver-specific ablation of TBL1 gene expression in healthy mice promotedhypertriglyceridemia and hepatic steatosis under both normal andhigh-fat dietary conditions. As TBL1 expression levels were found toalso inversely correlate with liver fat content in human patients, thelack of hepatic TBL1/TBLR1 cofactor activity may represent a molecularrationale for hepatic steatosis in subjects with obesity and themetabolic syndrome.

Berriel Diaz, M., et al. (Nuclear receptor co-factor RIP140 controlslipid metabolism during wasting in mice. 2008. Hepatology 48: 782-791)describe that by preventing the mobilization of hepatic TG stores, theinduction of RIP140 in liver provides a molecular rationale for hepaticsteatosis in starvation, sepsis, or cancer cachexia. Inhibition ofhepatic RIP140 transcriptional activity might, thereby, provide anattractive adjunct scheme in the treatment of these conditions.

Farese et al. (in: The problem of establishing relationships betweenhepatic steatosis and hepatic insulin resistance. Cell Metab. 2012 May2; 15(5):570-3) describe that excessive deposition of fat in the liver(hepatic steatosis) is frequently accompanied by hepatic insulinresistance.

Major classes of anti-diabetic and/or insulin sensitizing drugs includesulfonyl ureas, metformin, thiazolidine diones, alpha-glucosidaseinhibitors, incretin mimetics, and dipeptidylpeptidase 4 inhibitors, allof which are associated with severe limitations (for review see Moller,Metabolic disease drug discovery—“hitting the target” is easier saidthan done. Cell Metab. 2012 Jan. 4; 15(1):19-24).

Despite the key role of insulin resistance in the pathogenesis of type 2diabetes, effective and safe insulin sensitizers are still lacking.Indeed, current drugs of the thiazolidinedione family display a moderateefficacy profile and are accompanied by substantial side effects,including weight gain, increased risk of heart failure, possibleincreased risk of bladder cancer, and an increased risk for myocardialinfarction, e.g. leading to the recent market withdrawal ofrosiglitazone.

WO 2013/076501 discloses a screening method for identifying agentsuseful in the treatment and/or prevention of a disease associated withinsulin resistance and/or glucose intolerance which comprises the stepof investigating the capacity of a test agent to inhibit the Vps34signaling pathway and/or the RhoIota3Kappa-02beta signaling pathway.Similarly, WO 2005/059564 discloses a method for scanning molecules thatmodulate the activity of Retinol Binding Protein 4 (RBP4) and their usein treatment of insulin resistance are described. Also described aremethods of diagnosing insulin resistance and related conditions bydetecting modulation of RBP4 activity.

WO 2012/158123 relates to a method of treating or preventing insulinresistance syndrome in an animal body by administering an inhibitor ofprotein kinase RNA-like endoplasmic reticulum kinase (PERK) gene, or afunctional variant thereof, or an inhibitor of PERK protein or afunctional variant thereof or a method of reducing activity oftranscription factors of the FOXO family (Foxo 1, 3a, 4 and 6) byadministering an inhibitor of protein kinase RNA-like endoplasmicreticulum kinase (PERK) gene, or a functional variant thereof, or aninhibitor of PERK protein or a functional variant thereof.

WO 2014/202602 generally refers to modulators, in particular inhibitors,of TSC22D4 activity or expression and their uses for the prevention,treatment, and/or regulation of insulin resistance, metabolic syndromeand/or diabetes and/or for improving insulin sensitivity in a mammal. WO2014/202602 further relates to screening methods in order to identifythese modulators.

While the experimental knockdown by means of viral delivery ofTSC22D4-directed shRNA or miRNA constructs has been proven toefficiently improve the metabolic status of diabetic animals, siRNAconstructs suitable for the efficient and specific knockdown of TSC22D4in various species upon delivery by different technologies had not yetbeen identified.

In view of the above described flaws in the background art, theobjective of the present invention is to provide a new therapeuticstrategy to prevent, treat, and/or regulate insulin resistance,metabolic syndrome and/or diabetes and/or to improve insulinsensitivity.

In a first aspect of the present invention, the above objective issolved by providing an inhibitor of the expression and/or biologicalactivity of TSC22D4 selected from an oligonucleotide that is aninterfering ribonucleic acid, PNA (protein nucleic acid) or LNA (lockednucleic acid), comprising at least one the following sequences:5′-GGACGUGUGUGGAUGUUUAdTdT-3′ (SEQ ID No. 1);5′-GGAUGUUUACGAGAGAGAUdTdT-3′ (SEQ ID No. 2);5′-AGUCCCACCUCAUGUUUGCdTdT-3′ (SEQ ID No. 3); an antisense sequencethereof, or functional variants thereof.

mhD4-siRNA1: (NM_030935.3_siRNA_1024; ORF) (SEQ ID No. 1)Sense: 5′-GGACGUGUGUGGAUGUUUAdTdT-3′; (SEQ ID No. 4)Antisense: 5′-UAAACAUCCACACACGUCCdTdT-3′; GC: 47% (w/o TT-overhang)mD4-siRNA2: (NM_023910.6_siRNA_993; ORF) (SEQ ID No. 2)Sense: GGAUGUUUACGAGAGAGAUdTdT-3′; (SEQ ID No. 5)Antisense: AUCUCUCUCGUAAACAUCCdTdT-3′; GC: 42.1% (w/o TT-overhang)mhD4-siRNA3: (SEQ ID No. 3) Sense: 5′-AGUCCCACCUCAUGUUUGCdTdT-3′;(SEQ ID No. 6) Antisense: 5′-GCAAACAUGAGGUGGGACUdTdT-3′;GC: 52.6% (w/o TT-overhang)

Recently, the inventors have shown that transcriptional regulatortransforming growth factor beta1 stimulated clone 22 D4 (TSC22D4)controls hepatic and systemic insulin sensitivity. Liver specific lossof TSC22D4 significantly improved glucose tolerance and insulinsensitivity and counteracted hyperinsulinemia in wild-type mice.ChlP-Seq analysis of the TSC22D4 cistrome in combination with highthroughput TSC22D4 target transcriptome studies in healthy animalsrevealed that major nodes of the insulin signaling pathway were directlyor indirectly targeted by TSC22D4, most notably lipocalin 13.

Indeed, down-regulation or overexpression of TSC22D4 in primary mousehepatocytes as well as in wild-type mice led to the up- ordown-regulation of the intracellular insulin signaling pathway, asdetermined by phosphorylation of Akt/PKB kinase at Ser473 and ofGSK3beta at Ser9, in response to acute insulin exposure, respectively.Intriguingly, hepatic inactivation of TSC22D4 in diabetic db/db miceimproved glucose intolerance and insulin resistance in these animals andnormalized blood glucose to almost healthy levels. In congruence with anoverall improvement of the metabolic status in diabetic animals,circulating levels of pro-inflammatory cytokines and resistin weresignificantly lower in mice with liver-specific TSC22D4 deficiency.

While the experimental knockdown by means of viral delivery ofTSC22D4-directed shRNA or miRNA constructs has been proven toefficiently improve the metabolic status of diabetic animals, siRNAconstructs suitable for the efficient and specific knockdown of TSC22D4in various species upon delivery by different technologies had not yetbeen identified.

The inactivation of TSC22D4 in hepatoma cells did not increase cellulargrowth but rather decreased proliferation, suggesting that the insulinsensitizing function of TSC22D4 does not result in increased cancersusceptibility in affected cells/or organs. In addition, hepaticinactivation of TSC22D4 also did not cause hypoglycemia.

An “inhibitor” is a substance that can reduce the effectiveness of acatalyst in a catalyzed reaction (either a non-biological catalyst or anenzyme). An inhibitor referred to herein can reduce the effectiveness ofthe activity of an enzyme; also, an inhibitor referred to herein canreduce the effectiveness of the expression of an enzyme. In the contextof the present invention, a preferred inhibitor is an oligonucleotide.

The term “oligonucleotide” generally refers to an interferingribonucleic acid (IRNA), or protein nucleic acid (PNA) or locked nucleicacid (LNA). The term “oligonucleotide” generally refers to asingle-stranded nucleotide polymer made of more than 19 nucleotidesubunits covalently joined together. Preferably between 19 and 100nucleotide units are present, most preferably between 19 and 50nucleotides units are joined together, as also explained further below.

The sugar groups of the nucleotide subunits may be ribose, deoxyriboseor modified derivatives thereof such as 2′-0-methyl ribose. Thenucleotide subunits of an oligonucleotide may be joined byphosphodiester linkages, phosphorothioate linkages, methyl phosphonatelinkages or by other rare or non-naturally-occurring linkages that donot prevent hybridization of the oligonucleotide. Furthermore, anoligonucleotide may have uncommon nucleotides or non-nucleotidemoieties.

The term “oligonucleotide” may also refer, in the context of thespecification, to a nucleic acid analogue of those known in the art, forexample Locked Nucleic Acid (LNA), or a mixture thereof. The term“oligonucleotide” includes oligonucleotides composed of naturallyoccurring nucleobases, sugars and internucleoside (backbone) linkages aswell as oligonucleotides having non-naturally-occurring portions whichfunction similarly or with specific improved functions. A fully orpartly modified or substituted oligonucleotide is often preferred overnative forms because of several desirable properties of sucholigonucleotides such as for instance, the ability to penetrate a cellmembrane, good resistance to extra- and intracellular nucleases, highaffinity and specificity for the nucleic acid target. Methods ofmodifying oligonucleotides in this manner are known in the art.

In some oligonucleotides, sometimes called oligonucleotide mimetics,both the sugar and the internucleoside linkage, i.e., the backbone, ofthe nucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an oligonucleotide mimetic thathas been shown to have excellent hybridization properties, is referredto as a protein nucleic acid (PNA). In PNA compounds, the sugar-backboneof an oligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone.

A further modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugarring, thereby forming a bicyclic sugar moiety. The linkage is preferablya methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′carbon atom, wherein n is 1 or 2. The term “LNA” generally refers to anucleotide containing one bicyclic nucleoside analogue, also referred toas a LNA monomer, or an oligonucleotide containing one or more bicyclicnucleoside analogues.

Preferred is the inhibitor according to the present invention, whereinthe interfering ribonucleic acid is a small interfering ribonucleic acid(siRNA) or small hairpin ribonucleic acid (shRNA) or micro ribonucleicacid (miRNA) or combinations thereof.

Further preferred is the inhibitor according to the present invention,wherein the siRNA has a length of between 19 to 30 nucleotides.

In one aspect, the bioactive agent utilizes “RNA interference (RNAi)”.RNAi is a process of sequence-specific, post-transcriptional genesilencing initiated by double stranded RNA (dsRNA) or siRNA. RNAi isseen in a number of organisms such as Drosophila, nematodes, fungi andplants, and is believed to be involved in anti-viral defense, modulationof transposon activity, and regulation of gene expression. During RNAi,dsRNA or siRNA induces degradation of target mRNA with consequentsequence-specific inhibition of gene expression. As used herein, a“small interfering RNA” (siRNA) is a RNA duplex of nucleotides that istargeted to the gene of TSC22D4. A “RNA duplex” refers to the structureformed by the complementary pairing between two regions of a RNAmolecule. siRNA is “targeted” to a gene in that the nucleotide sequenceof the duplex portion of the siRNA is complementary to a nucleotidesequence of the targeted gene. In some embodiments, the length of theduplex of siRNAs is less than 30 nucleotides. In some embodiments, theduplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, thelength of the duplex is 19-25 nucleotides in length. The RNA duplexportion of the siRNA can be part of a hairpin structure. In addition tothe duplex portion, the hairpin structure may contain a loop portionpositioned between the two sequences that form the duplex. The loop canvary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11,12 or 13 nucleotides in length. The hairpin structure can also contain3′ and/or 5′ overhang portions. In some embodiments, the overhang is a3′ and/or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. ThesiRNA can be encoded by a nucleic acid sequence, and the nucleic acidsequence can also include a promoter. The nucleic acid sequence can alsoinclude a polyadenylation signal. In some embodiments, thepolyadenylation signal is a synthetic minimal polyadenylation signal.

As used herein, the term “siRNA” refers to a ribonucleic acid (RNA) orRNA analog comprising between about 19 to 50 nucleotides (or nucleotideanalogs) capable of directing or mediating the RNA interference pathway.These molecules can vary in length and can contain varying degrees ofcomplementarity to their target messenger RNA (mRNA) in the antisensestrand. The term “siRNA” includes duplexes of two separate strands, i.e.double stranded RNA, as well as single strands that can form hairpinstructures comprising of a duplex region. The siRNA may have a length ofbetween about 19 to 50 nucleotides, or between about 25 to 50nucleotides, or between about 30 to 50 nucleotides, or between about 35to 50 nucleotides, or between about 40 to 50 nucleotides. In oneembodiment, the siRNA has a length of between 19 to 30 nucleotides.

The application of siRNA to down-regulate the activity of its targetmRNA is known in the art. In some embodiments, mRNA degradation occurswhen the anti-sense strand, or guide strand, of the siRNA directs theRNA-induced silencing complex (RISC) that contains the RNA endonucleaseAgo2 to cleave its target mRNA bearing a complementary sequence.Accordingly, the siRNA may be complementary to any portion of varyinglengths on the PERK gene. The siRNA may also be complementary to thesense strand and/or the anti-sense strand of the TSC22D4 gene.Accordingly, siRNA treatment may be used to silence the TSC22D4 gene,thereby depleting the TSC22D4 protein downstream.

The term “shRNA”, as used herein, refers to a unimolecular RNA that iscapable of performing RNAi and that has a passenger strand, a loop and aguide strand. The passenger and guide strand may be substantiallycomplementary to each other. The term “shRNA” may also include nucleicacids that contain moieties other than ribonucleotide moieties,including, but not limited to, modified nucleotides, modifiedinternucleotide linkages, non-nucleotides, deoxynucleotides, and analogsof the nucleotides.

miRNAs down-regulate their target mRNAs. The term “miRNA” generallyrefers to a single stranded molecule, but in specific embodiments, mayalso encompass a region or an additional strand that is partially(between 10% and 50% complementary across length of strand),substantially (greater than 50% but less than 100% complementary acrosslength of strand) or fully complementary to another region of the samesingle-stranded molecule or to another nucleic acid. Thus, nucleic acidsmay encompass a molecule that comprises one or more complementary orself-complementary strand(s) or “complements” of a particular sequencecomprising a molecule. For example, precursor miRNA may have aself-complementary region, which is up to 100% complementary. miRNAprobes or nucleic acids of the invention can include, can be or can beat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% complementary to their target.

Most preferred is the inhibitor according to the present invention,wherein the siRNA consists of a sequence: according to SEQ ID No. 1 to 3an antisense sequence thereof.

Also preferred is the inhibitor according to the present invention,wherein the functional variant thereof comprises at least one modifiedor substituted nucleotide. The term “functional variant” also includes afragment, a variant based on the degenerative nucleic acid code or achemical derivative. A functional variant may have conservative changes,wherein a substituted nucleic acid has similar structural or chemicalproperties to the replaced nucleic acid. A functional variant may alsohave a deletion and/or insertion of one or more nucleic acids. It isunderstood that the functional variant at least partially retains itsbiological activity, e.g. function, of the TSC22D4 gene, or evenexhibits improved biological activity.

Examples of modified oligonucleotides include, but are not limited tooligonucleotides with phosphorothioate backbones (see above) andoligonucleosides with heteroatom backbones, and in particular—CH₂—NH—O—CH2-, —CH₂—N(CH₃)—O—CH₂—, —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—]. Also usable areoligonucleotides having morpholino backbone structures. Modifiedoligonucleotides used as interfering ribonucleic acids may also containone or more substituted sugar moieties. Preferred oligonucleotidescomprise one of the following at the 2′ position: OH; F; O—, S—, orN-alkyl; O—, S—, or N-alkenyl; O—, S— or N— alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl.Particular examples include, but are not limited toO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other exemplary oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.One exemplary modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE), i.e., an alkoxyalkoxygroup.

Another aspect then relates to a recombinant vector, comprising anoligonucleotide according to the present invention. Generally, theoligonucleotide is inserted into an expression vector, such as aplasmid, for expression. If necessary, the oligonucleotide may be linkedto the appropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host, although suchcontrols are generally available in the expression vector. The vector isthen introduced into the host through standard techniques.

Vectors that express siRNAs within mammalian cells typically use an RNApolymerase III promoter to drive expression of a short hairpin RNA thatmimics the structure of an siRNA. The insert that encodes this hairpinis designed to have two inverted repeats separated by a short spacersequence. One inverted repeat is complementary to the mRNA to which thesiRNA is targeted. A string of thymidines added to the 3′ end serves asa pol III transcription termination site. Once inside the cell, thevector constitutively expresses the hairpin RNA, which induces silencingof the target gene.

Other suitable vectors include viral vectors, such as adenoviral,retroviral and lentiviral viruses or the respective expression systems(see, for example Catanotto, D. et al. (2002) Functional siRNAexpression from transfected PCR products. RNA 8, 1454-1460; Barton, G.M. et al. (2002) Retroviral delivery of small interfering RNA intoprimary cells. Proc Natl Acad Sci USA. 99(23):14943-5. Abbas-Terki, T.et al. (2002) Lentiviral-mediated RNA interference. Hum. Gene Ther. 13,2197-2201, and Xia, H. et al. (2002) siRNA-mediate gene silencing invitro and in vivo. Nat. Biotechnol. 20, 1006-1010).

Generally, not all of the hosts will be transformed by the vector.Therefore, it will be necessary to select for transformed host cells.One selection technique involves incorporating into the expressionvector a DNA sequence, with any necessary control elements, that codesfor a selectable trait in the transformed cell, such as antibioticresistance. Alternatively, the gene for such selectable trait can be onanother vector, which is used to co-transform the desired host cell.Host cells that have been transformed by the oligonucleotide of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Other examples can be found in the literature, e.g. in Yang J. et al.(Design, preparation and application of nucleic acid delivery carriers.”Biotechnol Adv. 2014 July-August; 32(4):804-17).

Each of the classes of nucleic acids as described herein (e.g. theoligonucleotides and/or the vectors) can be introduced into cells by anumber of methods. In lipid-mediated transfection, cells take innon-covalent complexes between nucleic acid and a lipid or polymerreagent by endocytosis. Electroporation utilizes a brief electricalpulse to cause disruptions or holes in the cells' plasma membranethrough which nucleic acid enters. Both of these methods successfullydeliver any of the RNAi nucleic acids expect viral vectors. Viral vectordelivery only occurs by infection of cells with the corresponding virus,usually using helper viruses. Infection of the desired cell line withvirus introduces the siRNA or shRNA and knocks down gene expression.

Another aspect of the present invention then relates to a recombinantcell, preferably a recombinant hepatocytic cell, comprising anoligonucleotide according to the present invention, or a recombinantvector according to the present invention. A “cell” according to theinvention can be a prokaryotic or eukaryotic cell. A “cell” according tothe invention is preferably, and without being limited to it, selectedfrom liver cells. Mammalian cells may be preferably selected from ahuman, rabbit, mouse or rat. Preferably, the cell is a human cell, e.g.a hepatocytic cell. The term “cell” also includes cells of an animalmodel. Also, a cell can be part of a tissue culture.

The object of the invention is also solved by a method for producing apharmaceutical composition, comprising the steps of formulating said atleast one inhibitor according to the present invention with at least onepharmaceutically acceptable excipient. The carrier and/or excipient ofthe pharmaceutical composition must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

Another aspect of the present invention then relates to a pharmaceuticalcomposition, comprising at least one of the inhibitor according to thepresent invention, the recombinant vector according to the presentinvention, and the recombinant cell according to the present invention,together with a pharmaceutically acceptable carrier. Preferred is apharmaceutical composition according to the present invention, whereinsaid pharmaceutical composition is for administration orally, rectally,transmucosally, transdermally, intestinally, parenterally,intramuscularly, intrathecally, direct intraventricularly,intravenously, intraperitoneally, intranasally, intraocularly, orsubcutaneously.

Another aspect of the present invention then relates to the inhibitoraccording to the present invention, the expression vector according tothe present invention, the recombinant cell according to the presentinvention or the pharmaceutical composition according to the presentinvention for use in the prevention, regulation, and/or treatment ofdiseases.

Insulin resistance syndrome makes up a broad clinical spectrum and isdefined as any abnormalities associated with insulin resistance.Abnormalities such as the resistance to insulin, diabetes, hypertension,dyslipidemia and cardiovascular disease constitute the insulinresistance syndrome.

The insulin resistance syndrome may be diet-induced insulin resistanceand/or obesity-induced insulin resistance. Diet-induced insulinresistance means that the resistance to insulin is induced by a diethigh in saturated fat and carbohydrates. Obesity-induced insulinresistance means that the resistance to insulin is induced by a geneticpredisposition to obesity or obesity which is due to dietary habits.

Another aspect of the present invention thus relates to the inhibitoraccording to the present invention, the expression vector according tothe present invention, the recombinant cell according to the presentinvention or the pharmaceutical composition according to the presentinvention for use in the prevention, regulation, and/or treatment of adisease that is selected from insulin resistance, hypertension,dyslipidemia, coronary artery disease, metabolic syndrome and/ordiabetes type 1 or 2, and/or for improving insulin sensitivity, such as,for example, insulin sensitivity in the context of a tumorous disease.Preferably, the insulin resistance syndrome is diet-induced insulinresistance and/or obesity-induced insulin resistance.

The object is further solved by a method for treating and/or preventinga disease selected from insulin resistance, metabolic syndrome and/ordiabetes in a subject in need thereof, comprising the step ofadministering an effective amount of an inhibitor according to thepresent invention or the pharmaceutical composition according to thepresent invention to said patient in need thereof.

The disclosed methods may be used for treating any one of the followingconditions which are caused by insulin resistance syndrome: insulinresistance, hypertension, dyslipidemia, Type 2 diabetes or coronaryartery disease.

The term “prevention” in the context of the present invention shall beunderstood as a medical intervention which aims to avoid the occurrenceof a negative event which most likely leads to the worsening of thecondition of a patient having a disease, or to the injury or the deathof a healthy and/or ill subject. The “patient in need thereof” can be,without being limited to it, any animal or human suffering from adisease related to insulin resistance syndrome, especially insulinresistance, hypertension, dyslipidemia, Type 2 diabetes or coronaryartery disease. Preferably, the subject in need thereof is a human.

The object is further solved by a therapeutic kit, comprising theinhibitor according to the present invention, the recombinant vectoraccording to the present invention, the recombinant cell according tothe present invention or the pharmaceutical composition according to thepresent invention, optionally together with suitable buffers andexcipients, and instructions for use.

The object is further solved by a therapeutic kit according to thepresent invention for use in the prevention, regulation, and/ortreatment of a disease, wherein said disease is selected from insulinresistance, hypertension, dyslipidemia, coronary artery disease,metabolic syndrome and/or diabetes type 1 or 2, and/or for improvinginsulin sensitivity, such as, for example, insulin sensitivity in thecontext of a tumorous disease.

Recently, the inventors have shown that transcriptional regulatortransforming growth factor beta 1 stimulated clone 22 D4 (TSC22D4)controls hepatic and systemic insulin sensitivity. Liver specific lossof TSC22D4 significantly improved glucose tolerance and insulinsensitivity and counteracted hyperinsulinemia in wild-type mice.ChlP-Seq analysis of the TSC22D4 cistrome in combination with highthroughput TSC22D4 target transcriptome studies in healthy animalsrevealed that major nodes of the insulin signaling pathway were directlyor indirectly targeted by TSC22D4, most notably lipocalin 13. Indeed,down-regulation or over-expression of TSC22D4 in primary mousehepatocytes as well as in wild-type mice led to the up- ordown-regulation of the intracellular insulin signaling pathway, asdetermined by phosphorylation of Akt/PKB kinase at Ser473 and ofGSK3beta at Ser9, in response to acute insulin exposure, respectively.

Intriguingly, hepatic inactivation of TSC22D4 in diabetic db/db miceimproved glucose intolerance and insulin resistance in these animals andnormalized blood glucose to almost healthy levels. In congruence with anoverall improvement of the metabolic status in diabetic animals,circulating levels of pro-inflammatory cytokines and resistin weresignificantly lower in mice with liver-specific TSC22D4 deficiency.

Inactivation of TSC22D4 in hepatoma cells did not increase cellulargrowth but rather decreased proliferation, suggesting that the insulinsensitizing function of TSC22D4 does not result in increased cancersusceptibility in affected cells/or-a ans. In addition, hepaticinactivation of TSC22D4 also did not cause hypoglycemia.

While the experimental knockdown by means of viral delivery ofTSC22D4-directed shRNA or miRNA constructs has been proven toefficiently improve the metabolic status of diabetic animals, siRNAconstructs suitable for the efficient and specific knockdown of TSC22D4in various species upon delivery by different technologies had not yetbeen identified. In order to overcome this problem, the inventors haveidentified, functionally tested and validated various siRNAs directedagainst the TSC22D4 mRNA sequence in in vitro knockdown studies usingmurine Hepa1.6 as well as human Huh7 hepatoma cells as disclosed herein.

The following figures, sequences, and examples merely serve toillustrate the invention and should not be construed to restrict thescope of the invention to the particular embodiments of the inventiondescribed in the examples. For the purposes of the present invention,all references as cited in the text are hereby incorporated in theirentireties.

FIG. 1 shows the knockdown efficiency of color-coded, selectedTSC22D4-directed siRNAs in murine hepatoma cells. Relative mRNA levelsare shown. All other tested siRNA sequences did not show any significantTSC22D4 knockdown in these experiments (not shown).

FIG. 2 shows the knockdown efficiency of mhD4-siRNA1 upon transfectioninto murine Hepa1-6 hepatoma cells towards murine TSC22D4. Relative mRNAlevels are shown.

FIG. 3 shows the knockdown efficiency of mhD4-siRNA1 upon transfectioninto human Huh7 hepatoma cells towards human TSC22D4. Relative mRNAlevels are shown.

Sequence ID NOs. 1 to 6 show oligonucleotide sequences according to thepresent invention.

EXAMPLES

Recombinant Viruses

Adenoviruses expressing a TSC22D4 or a non-specific shRNA under thecontrol of the U6 promoter, or the TSC22D4 cDNA under the control of theCMV promoter were cloned using the BLOCK-iT Adenoviral RNAi expressionsystem (Invitrogen, Karlsruhe, Germany). Viruses were purified by thecesium chloride method and dialyzed against phosphate-buffered-salinebuffer containing 10% glycerol prior to animal injection, as describedpreviously (Herzig S, Hedrick S, Morantte I, Koo S H, Galimi F, MontminyM. CREB controls hepatic lipid metabolism through nuclear hormonereceptor PPAR-gamma. Nature. 2003; 426: 190-193. Herzig S, Long F, JhalaU S, Hedrick S, Quinn R, Bauer A, Rudolph D, Yoon C, Puigserver P,Spiegelman B, et al. CREB regulates hepatic gluconeogenesis through thecoactivator PGC-1. Nature. 2001; 413: 179-183). AAVs encoding control orTSC22D4-specific miRNAs under the control of a hepatocyte-specificpromoter were established as described previously (Rose A J, Frosig C,Kiens B, Wojtaszewski J F, Richter E A. Effect of endurance exercisetraining on Ca2+ calmodulin-dependent protein kinase II expression andsignaling in skeletal muscle of humans. J Physiol. 2007; 583: 785-795).

Animal Experiments

Male 8-12 week old C57Bl/6 and 10 week old db/db mice were obtained fromCharles River Laboratories (Brussels, Belgium) and maintained on a 12 hlight-dark cycle with regular unrestricted diet. Prior to insulin andglucose tolerance tests, animals were fasted for 4 h. Otherwise, animalswere fed ad libitum and had free access to water. For adenovirusinjections, 1-2×10⁹ plaque-forming units (pfu) per recombinant viruswere administered via tail vein injection. For AAV experiments, 5×10¹¹viruses were injected via the tail vein. In each experiment, 6-12animals received identical treatments and were analyzed under fasted (18hrs fasting), random fed or fed (18 hrs fasting followed by 6 hrsre-feeding) conditions as indicated. Organs including liver, epididymaland abdominal fat pads, and gastrocnemius muscles were collected afterspecific time periods, weighed, snap-frozen and used for furtheranalysis. Total body fat content was determined by an Echo MRI bodycomposition analyzer (Echo Medical Systems, Houston, USA). Animalhandling and experimentation was done in accordance with NIH guidelinesand approved by local authorities.

For the insulin tolerance tests a stock solution of 1 U Insulin/mL wasprepared using 0.9% sodium chloride. Mice were fasted for 4 hours priorto the experiment. The body weight of all animals was determined and theblood glucose levels were measured by cutting the tail with a razorblade. The blood drop was put onto a glucometer strip and measured. 1 Uinsulin/kg body weight was injected to C57B¹/6 and 1.5 U insulin/kg bodyweight was injected to db/db mice intraperitoneally. The blood glucoselevels were monitored after 20, 40, 60, 80 and 120 min.

For the glucose tolerance tests a stock solution of 20% glucose wasprepared using 0.9% sodium chloride. Mice were fasted for 4 hours priorto the experiment. The body weight of all animals was determined and theblood glucose levels were measured by cutting the tail with a razorblade. The blood drop was put onto a glucometer strip and measured. 54per gram of 20% glucose solution was injected to C57B¹/6 and db/db miceintraperitoneally. The blood glucose levels were monitored after 20, 40,60, 80 and 120 min.

Quantitative Taqman RT-PCR

Total RNA was extracted from homogenized mouse liver or cell lysatesusing Qiazol reagent (Qiagen, Hilden, Germany). cDNA was prepared byreverse transcription using the M-MuLV enzyme and Oligo dT primer(Fermentas, St. Leon-Rot, Germany). cDNAs were amplified usingassay-on-demand kits and an ABIPRISM 7700 Sequence detector (AppliedBiosystems, Darmstadt, Germany). RNA expression data was normalized tolevels of TATA-box binding protein (TBP) RNA.

Human TSC22D4 mRNA expression was measured by quantitative real-timeRT-PCR in a fluorescent temperature cycler using the TaqMan assay, andfluorescence was detected on an ABI PRISM 7000 sequence detector(Applied Biosystems, Darmstadt, Germany). Total RNA was isolated usingTRIzol (Life technologies, Grand Island, N.Y.), and 1 μg RNA was reversetranscribed with standard reagents (Life Technologies, Grand Island,N.Y.). From each RT-PCR, 2 μl were amplified in a 26 μl PCR reactionusing the Brilliant SYBR green QPCR Core reagent kit from stratagene (LaJolla, Calif.) according to the manufacturer's instructions.

Samples were incubated in the ABI PRISM 7000 sequence detector for aninitial denaturation at 95° C. for 10 min, followed by 40 PCR cycles,each cycle consisting of 95° C. for 15 s, 60° C. for 1 min and 72° C.for 1 min. Human TSC22D4 and Obp2a (LCN13) (determined by Hs00229526_m1and Hs01062934_g1, respectively) (Applied Biosystems, Darmstadt,Germany) mRNA expression was calculated relative to the mRNA expressionof hypoxanthine phosphoribosyltransferase 1 (HPRT1), determined by apremixed assay on demand for HPRT1 (Hs01003267_m1) (Applied Biosystems,Darmstadt, Germany). Amplification of specific transcripts was confirmedby melting curve profiles (cooling the sample to 68° C. and heatingslowly to 95° C. with measurement of fluorescence) at the end of eachPCR. The specificity of the PCR was further verified by subjecting theamplification products to agarose gel electrophoresis.

Protein Analysis

Protein was extracted from frozen organ samples or cultured hepatocytesin cell lysis buffer (Rose A J, Frosig C, Kiens B, Wojtaszewski J F,Richter E A. Effect of endurance exercise training on Ca2+calmodulin-dependent protein kinase II expression and signaling inskeletal muscle of humans. J Physiol. 2007; 583: 785-795) and 20 μg ofprotein were loaded onto 4-12% SDS-polyacrylamide gels and blotted ontonitrocellulose membranes. Western blot assays were performed asdescribed (Herzig et al, 2001) using antibodies specific for TSC22D4(Abcam, Cambridge, UK or Sigma, Munich, Germany), AKT, p-AKT, GSK, p-GSK(Cell signaling, Danvers, USA) or VCP (Abcam).

Plasmids and RNA Interference

For shRNA experiments, oligonucleotides targeting mouse and TSC22D4 (SEQID No. 1 to 3), were cloned into the pENTR/U6 shRNA vector (Invitrogen).

Cell Culture and Transient Transfection Assays

Primary mouse hepatocytes were isolated and cultured as described(Klingmuller U, Bauer A, Bohl S, Nickel P J, Breitkopf K, Dooley S,Zellmer S, Kern C, Merfort I, Sparna T, et al. Primary mouse hepatocytesfor systems biology approaches: a standardized in vitro system formodelling of signal transduction pathways. IEE Proc Syst Biol. 2006;153: 433-447). Briefly, male 8-12 week old C57B¹/6 mice wereanaesthetized by i.p. injection of 100 mg/kg body weight ketaminehydrochloride and 5 mg/kg body weight xylazine hydrochloride. Afteropening the abdominal cavity, the liver was perfused at 37° C. withHANKS I (8 g NaCl, 0.4 g KCl, 3.57 g Hepes, 0.06 g Na₂HPO₄×2 H₂O, 0.06 gKH₂PO₄ in 1 L distilled H₂O, 2.5 mM EGTA, 0.1% glucose, adjusted to pH7.4) via the portal vein for 5 min and subsequently with HANKS II (8 gNaCl, 0.4 g KCl, 3.57 g Hepes, 0.06 g Na₂HPO₄×2 H₂O, 0.06 g KH₂PO₄ in 1L distilled H₂O, 0.1% glucose, 3 mg/ml collagenase CLSII, 5 mM CaCl₂,adjusted to pH 7.4) for 5-7 min until disintegration of the liverstructure was observed. The liver capsule was removed and the cellsuspension was filtered through a 100 μm mesh. The cells were washedand, subsequently, viability of cells was determined by trypan bluestaining. 1 000 000 living cells/well were seeded on collagen I-coatedsix-well plates. After 24 h, cells were infected with recombinantadenoviruses at a multiplicity of infection of 100. For stimulationexperiments, primary hepatocytes were treated with PBS (control medium)or insulin at a concentration of 100 nM/6-well for 10 minutes. Cellswere harvested 48 h after infection.

Cistrome Analysis of Hepatic TSC22D4

KEGG-Pathway analysis of Chip-Sequencing results were sorted bysignificance. The Insulin signaling pathway was found to besignificantly regulated (p=0.00005). Chip-Sequencing was performed inliver extracts from Flag-TSC22D4 cDNA adenovirus-injected male C57Bl/6mice 7 days after injection.

Results

The sequences worked very efficiently for both mouse and human TSC asseen in 4 independent experiments (see Figures). There is a nonspecificdTdT overhang attached to each sequence. The sequences matched both themouse and the human TSC sequence to 100%.

Based on these results, the sequences according to the present invention(SEQ ID No. 1 to 3) were chosen as primary candidates to be used fortherapeutic purposes as it shows a superior knockdown efficiency towardsTSC22D4 and targets a variety of species, including mouse, non-humanprimates and humans. The sequences were identified, functionally testedand validated various siRNAs directed against the TSC22D4 mRNA sequencein in vitro knockdown studies using murine Hepa1.6 as well as human Huh7hepatoma cells. In particular the mhD4-siRNA1 showed a superiorknockdown efficiency towards TSC22D4 and targets a variety of species,including mouse, non-human primates and humans.

mhD4-siRNA1: (NM_030935.3_siRNA_1024; ORF) (SEQ ID No. 1)Sense: 5′-GGACGUGUGUGGAUGUUUAdTdT-3′; (SEQ ID No. 4)Antisense: 5′-UAAACAUCCACACACGUCCdTdT-3′; GC: 47% (w/o TT-overhang)mD4-siRNA2: (NM_023910.6_siRNA_993; ORF) (SEQ ID No. 2)Sense: GGAUGUUUACGAGAGAGAUdTdT-3′; (SEQ ID No. 5)Antisense: AUCUCUCUCGUAAACAUCCdTdT-3′; GC: 42.1% (w/o TT-overhang)mhD4-siRNA3: (SEQ ID No. 3) Sense: 5′-AGUCCCACCUCAUGUUUGCdTdT-3′;(SEQ ID No. 6) Antisense: 5′-GCAAACAUGAGGUGGGACUdTdT-3′;GC: 52.6% (w/o TT-overhang)

The invention claimed is:
 1. An inhibitor of expression and/orbiological activity of transforming growth factor beta1 stimulated clone22 D4 (TSC22D4) wherein said inhibitor is selected from anoligonucleotide that is an interfering ribonucleic acid, PNA (proteinnucleic acid) or LNA (locked nucleic acid), and wherein saidoligonucleotide comprises at least one sequence selected from:5′-GGAUGUUUACGAGAGAGAUdTdT-3′ (SEQ ID NO: 2);5′-AGUCCCACCUCAUGUUUGCdTdT-3′ (SEQ ID NO: 3); and complementarysequences thereof.
 2. The inhibitor according to claim 1, wherein theinterfering ribonucleic acid is a small interfering ribonucleic acid(siRNA) or small hairpin ribonucleic acid (shRNA) or micro ribonucleicacid (miRNA) or a combination thereof.
 3. The inhibitor according toclaim 2, wherein the siRNA has a length of 19 to 30 nucleotides.
 4. Theinhibitor according to claim 2, wherein the siRNA consists of at leastone of SEQ ID NOs: 2 and
 3. 5. A recombinant vector, comprising anoligonucleotide according to claim
 1. 6. A recombinant cell, comprisingan oligonucleotide according to claim
 1. 7. A pharmaceuticalcomposition, comprising at least one of the inhibitor according to claim1, together with a pharmaceutically acceptable carrier.
 8. Thepharmaceutical composition according to claim 7, wherein saidpharmaceutical composition is formulated for administration orally,rectally, transmucosally, transdermally, intestinally, parenterally,intramuscularly, intrathecally, direct intraventricularly,intravenously, intraperitoneally, intranasally, intraocularly, orsubcutaneously.
 9. A method for prevention, regulation, and/or treatmentof a disease, and/or for improving insulin sensitivity, wherein saidmethod comprises administering, to a subject in need of such prevention,regulation, treatment, and/or improvement, an inhibitor of TSC22D4wherein said inhibitor is selected from an oligonucleotide that is aninterfering ribonucleic acid, PNA (protein nucleic acid) or LNA (lockednucleic acid), and wherein said oligonucleotide comprises at least onesequence selected from: 5′-GGACGUGUGUGGAUGUUUAdTdT-3′ (SEQ ID NO: 1);5′-GGAUGUUUACGAGAGAGAUdTdT-3′ (SEQ ID NO: 2);5′-AGUCCCACCUCAUGUUUGCdTdT-3′ (SEQ ID NO: 3); and complementarysequences thereof.
 10. The method, according to claim 9, wherein saiddisease is selected from insulin resistance, hypertension, dyslipidemia,coronary artery disease, metabolic syndrome and diabetes type
 1. 11. Themethod, according to claim 9, wherein the insulin resistance isdiet-induced insulin resistance and/or obesity-induced insulinresistance.
 12. A therapeutic kit, comprising the inhibitor according toclaim 1, optionally together with suitable buffers and excipients, andinstructions for use.
 13. The therapeutic kit according to claim 12 withinstructions for use in the prevention, regulation, and/or treatment ofa disease, wherein said disease is selected from insulin resistance,hypertension, dyslipidemia, coronary artery disease, metabolic syndromeand/or diabetes type 1 or 2, and/or for improving insulin sensitivity.14. The method, according to claim 9, for improving insulin sensitivityin the context of a tumorous disease.
 15. The inhibitor according toclaim 1, wherein the oligonucleotide comprises SEQ ID NO: 2 or thecomplementary sequence thereof.
 16. The inhibitor according to claim 1,wherein the oligonucleotide comprises SEQ ID NO: 3 or the complementarysequence thereof.
 17. The inhibitor according to claim 1, wherein theinterfering ribonucleic acid is a siRNA.
 18. The inhibitor according toclaim 17, wherein the siRNA is a RNA duplex comprising at least oneof: 1) SEQ ID NO: 2 and SEQ ID NO: 5; and 2) SEQ ID NO: 3 and SEQ ID NO:6.